Molecular cloning and physiological effects of brushtail possum interleukin-1β

Interleukin-1β (IL-1β) was isolated from LPS-stimulated brushtail possum alveolar macrophages using PCR primers based on conserved regions of mammalian IL-1β. The complete cDNA was cloned by 5′ and 3′ rapid amplification of cDNA ends (RACE). The predicted protein of 269 amino acids shared 43–46% identity with several mammalian IL-1β proteins. Constructs were made to express the mature IL-1β in Escherichia coli and two recombinant IL-1β proteins, rpIL-1β1 and rpIL-1β2, which differed in length by four amino acids at the N-terminus, were produced. Both proteins induced a weak proliferative response in a possum thymocyte assay. Possums injected intravenously with 100 μg of rpIL-1β1 or rpIL-1β2 showed profound changes in body temperature and numbers of circulating leukocytes. A sharp decrease in temperature occurred within 2 h of administration followed by an elevation of temperature peaking at 24 h. The smaller rpIL-1β1 protein had a greater effect on temperature than rpIL-1β2. Both rpIL-1β proteins caused a marked decrease in number of neutrophils and lymphocytes at 2–6 h after injection. At 24 h after injection, neutrophil and lymphocyte numbers were elevated 6.0-fold and 2.6-fold, respectively in the possums injected with rpIL-1β1 and 3.9-fold and 1.5-fold, respectively in the possums injected with rpIL-1β2. Fibrinogen levels were elevated at 24 and 72 h after injection with both proteins. In comparison, neither recombinant bovine IL-1β (rbIL-1β) nor PBS had significant effects on body temperature or blood haematology. The studies have shown that the two recombinant forms of IL-1β were biologically active in possums and that the IL-1β with four fewer amino acids at the N-terminus was the more active.